Tissue Slice Staining

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Gilligan
Gilligan's picture
Tissue Slice Staining

I am trying to do alcian blue and Safranin staining of tissue slices.  This is my basic protocol...

dewax (60 degree oven for 30 min)
3x xylene soaks(5 min each)
rehydration
            100% ethanol 3 min
            100% ethanol 3 min
            90% ethanol 3 min
            70% ethanol 3 min

3x wash PBS @ pH1.0 (5 min)

Stain in 0.5% Alcian in PBS pH1.0 30 minutes
3x PBS washes (not 5 minutes here, just dip and remove)
Stain in 0.5% Safranin O in PBS 1.0 30 minutes
3x PBS washes (not 5 minutes here, just dip and remove)

Dehydration
            70% ethanol dip
            90% ethanol dip
            1000% ethanol dip

Mounting
    3x xylene 5 min

mount in depex

..........

This works for our cell suspensions but not for our tissue slices!  I guess the stain just washes over the tissue?   Any ideas of how to resolve this?  Longer stain times?  Has anyone else stained tissue slices before?

THANKS!

Arvind Singh Pundir
Arvind Singh Pundir's picture
 Dear Gilligan,

 Dear Gilligan,
your protocol seems to be right except during dehydration after staining instead if going to 70% alcohol go to directly 95% alcohol 2 changes and then 100% alcohol 3 changes, xylene 3 changes then mount with DPX,
as in 70 % alcohol your stain will come out so avoid that.....

hope it helps